Stress and aging can promote protein misfolding and aggregation, leading to cytotoxicity and disease. Indeed, protein misfolding and aggregation are linked with several intractable neurodegenerative diseases, including frontotemporal dementia (FTD). In ~45% of FTD cases, a nuclear RNA-binding protein with a prion-like domain, TDP-43, mislocalizes to cytoplasmic aggregates in degenerating neurons. We propose that a key therapeutic innovation for FTD (and other TDP-43 proteinopathies) will be to develop therapeutic TDP- 43 disaggregases that reverse the aberrant cytoplasmic aggregation of TDP-43 and return functional TDP-43 to the nucleus. We identified the first known human disaggregase system, which consists of three classes of human chaperones Hsp110 (e.g. Apg-2), Hsp70 (e.g. Hsc70), and Hsp40 (e.g. Hdj1) that act in concert. However, humans have multiple versions of Hsp110s (11 variants), Hsp70s (11 variants), and Hsp40s (52 variants) and the precise combinatorial interactions between Hsp110/Hsp70/Hsp40 variants are hypothesized to dictate substrate specificity. The three-gene nature of the human disaggregase system is a major challenge for engineering potentiated variants since most genetic engineering techniques target mutations to only a single gene. In addition, an exhaustive study of the additional Hsp110/Hsp70/Hsp40 complexes has been prohibitive since there are 6,292 possible Hsp110/Hsp70/Hsp40 combinations. This proposal will solve these challenges by leveraging the power of yeast genetics along with major advancements in synthetic DNA assembly and genome engineering technologies to explore and engineer human disaggregase systems in models of FTD. We hypothesize that a specific combination of Hsp110/Hsp70/Hsp40 most potently disaggregates TDP-43. Moreover, we hypothesize that it is possible to engineer and evolve potentiated variants of Hsp110, Hsp70, and Hsp40 to more effectively reverse deleterious TDP-43 misfolding in FTD. Thus, we will pursue four specific aims: (1) Define natural human Hsp110/Hsp70/Hsp40 combinations that rescue TDP-43 toxicity in yeast; (2) Engineer Hsp110/Hsp70/Hsp40 to rescue TDP-43 toxicity in yeast; (3) Define optimal TDP-43 disaggregases in vitro; and (4) Define optimal TDP-43 disaggregases that rescue FTD- linked TDP-43 toxicity in neuronal models. This experimental pipeline leverages the scale and power of yeast genetics to identify Hsp110/Hsp70/Hsp40 combinations and mutants that exhibit rescue of TDP-43 toxicity, which are then experimentally validated in bona fide human neurons. This project will greatly enhance our understanding of human disaggregase mechanisms by exhaustively screening the combinatorial space of three-gene disaggregase interactions and will likely identify new mechanisms to treat FTD.